Fig. 5. Western blot analysis of CFTR, NKCC and Na⁺,K⁺-ATPase in opercular epithelium (20 µg) and gill tissue (20 µg for CFTR and NKCC, 10 µg for Na⁺,K⁺-ATPase) from SW-adapted killifish. Proteins were separated on a 7.0 % polyacrylamide gel, transferred onto an Immobilion-P membrane and probed with anti-human CFTR monoclonal antibody (1.0 µg ml—1). Proteins were visualized following incubation in BCIP/NBT Blue substrate development solution. Lanes 1, 2 and 3 show heart (H) (negative control), opercular epithelium (Oe) and gill tissue (G), respectively. The anti-hCFTR antibody is specific for a protein of approx. 150 kDa and two lower molecular mass bands in the opercular epithelium and in the gill. Lanes 4, 5 and 6 show the same tissues probed with the NKCC T4 antibody with the main band at approx. 150 kDa and two lower molecular mass bands. Lanes 7, 8 and 9 show the same tissues probed with the Na⁺,K⁺-ATPase 5 antibody. Visible proteins appear at approx. 120 kDa (major band) and 101, 72 and 68 kDa. Molecular mass markers (kDa) were: 205, 116, 97.4, 84, 66, 55, 45 and 33 (from top to bottom). For means and S.E.M. (N=3), see text.